Heparin is a naturally occurring polyanionic polysaccharide, consisting of alternating uronic acids and glucosamines. Dependent on the length of the saccharide chain, heparin occurs with molecular weights of 6.000 to 30.000 g/mol. It is naturally produced in mast cells and basophile granulocytes to function as the bodies own coagulants. Most importantly, Heparin reduces blood clotting by activating antithrombin, inhibiting blood clotting factors such as Xa and Ila and by inhibiting thrombocyte aggregation.
Pharmacologically, heparin is used for preventing thrombosis or embolism in the course of surgeries, for reducing thrombotic or embolic reactions associated with extracorporeal cardiovascular systems and for treating acute phase heart insufficiency. Due to the high molecular weight and the anionic properties, heparin must be applied intravenously or subcutaneously. Heparin acts immediately upon administration, but, because it is digested rapidly within the organism, its effect lasts only for a few hours.
As a pharmaceutical, heparin is provided as unfractionated heparin and as low molecular weight heparin (LMWH). The unfractionated heparin is obtained from animal intestine mucosa or lung and mostly contains long chain molecules which are digested rapidly. Moreover, only 30% of the unfractionated heparin is pharmaceutically effective. LMWH, in contrast, is obtained by restricted digestion of native heparin, it is digested slower within the organism and contains a higher fraction of pharmacologically effective molecules. However, it cannot be antagonized by administration of antidotes such as protamine sulfate, bearing the risk of bleedings resulting from over dosage. Thus, to maintain constant heparin levels of unfractionated or low molecular weight heparin, within the body, that are sufficiently inhibiting blood clotting without causing bleedings or other adverse reactions, such as heparin induced thrombocytopenia, regular monitoring of the concentration of heparin in the blood is required.
Commonly, indirect methods as e.g. the prothrombin time method or the factor Xa activity test are used to determine blood clotting conditions. However, these methods do not detect heparin itself and can be influenced by factors other than heparin (Despotis et al., 1999). Some of the established test systems, as e.g. the prothrombin time method are applicable to unfractionated heparin only. Moreover, these methods are time consuming and require laboratory equipment. Thus they are particularly unsuited for emergency medical aid or point-of-care applications. Similar, also electrochemical methods, such as polymer membrane based ion-selective electrodes or ion-selective field effect transistors are elaborate and susceptible to influences of molecules other than heparin.
Recently, methods have been developed, e.g optical tests that rely on color reactions, to detect heparin. The chemical dyes, that have been found to interact with heparin so far, show emission at wavelengths within or close to the blood owns fluorescent and in addition their emission responses are greatly reduced in the presence of blood serum or plasma (Wright et al., 2005; Wang et al., 2008). These side effects may result from strong interference of the serum/plasma matrix with the chemical dyes, leading to a competitive binding of the dye to heparin or matrix molecules. The resulting low signal to background ratio hinders the accurate detection of heparin in blood or blood derived samples. Therefore a fast and easy method to detect heparin is required that also shows a low susceptibility to interfere with matrix molecules of blood, to assure the assays reliability.